Arginine deiminase is an enzyme which catalyzes the irreversible hydrolysis of arginine, an essential amino acid, to citrulline and ammonia. Arginine deiminase, a type of arginase, has been found in a number of species, both prokaryotic and eukaryotic. Arginases have been identified in liver cells, skin cells, bacteria such as streptococci, yeasts, and in various mycoplasmas. Some species of mycoplasmas use arginine deiminase as part of their energy generating metabolism and, as such, are good sources of this enzyme.
Mycoplasmas represent the simplest form of a self-replicating organism. They are small, bacteria-like microorganisms which, unlike bacteria and other prokaryotic cells, lack a cell wall. Their genome size, which is about 5.times.10.sup.8 daltons, is approximately one sixth that of Escherichia coli. Mycoplasmas inhabit the alimentary, respiratory and genito-urinary tracts of humans and animals and are a common contaminant of cell cultures.
Two major pathways exist for the generation of energy in mycoplasmas. One is classical glycolysis, wherein glucose is broken down into pyruvic acid and ATP. The second is the utilization of arginine. In this second pathway, arginine deiminase converts arginine to citrulline, and ultimately to NH.sub.3, CO.sub.2 and ATP. This mechanism is illustrated below: ##STR1## Mycoplasmas may be classified into arginine-positive or arginine-negative organisms. Most arginine-positive species do not ferment glucose and most fermenters do not hydrolyze arginine. Some, however, metabolize both. Barille (In: Methods in Mycoplasmology, Vol. 1, 1983, pp. 345-448).
Mycoplasmas which use arginine as an energy source (arginine positive), typically have large amounts of arginine deiminase in their cytoplasm. Schimke et al. (J. Biol. Chem. (1966) 241:2228-2236) found that the enzyme constitutes about 10% of the cytoplasmic protein of Mycoplasma arthritidis. As such, and as indicated above, mycoplasmas using arginine deiminase as part of their energy generating metabolism are good sources of enzyme.
Some species of mycoplasma are pathogenic to humans, the most notable being arginine-negative M. pneumoniae, which produces pneumonia. Other species of mycoplasma produce diseases in laboratory, farm and domestic animals, citrus plants and other crops, and in insects.
Two of three species from which arginine deiminase has been routinely prepared, M. orale and M. salivarium, are non-pathogenic, normal human flora. The third, M. arginini is a bovine isolate.
As mentioned above, mycoplasmas are common contaminants of cell culture. They produce a diversity of effects in cell culture including changes in viral titers, induction of interferon, mitogenesis, induction of cytokines, chromosomal aberrations, and activation of macrophages.
Mycoplasmal arginine deiminases are 1000 times more potent than mammalian arginases in inhibiting the growth of human tumor cells in vitro. Miyazaki et al. (Ca. Res. (1990) 50:4522-4527). Bach et al. (Br. J. Ca. (1965) 19:379-384). However, their development as anti-neoplastic agents and any unfavorable effects in vivo remain to be thoroughly explored.
Anti-neoplastic agents which inhibit the metabolism of rapidly proliferating cells has been the mainstay in cancer chemotherapy. Most chemotherapeutic drugs (e.g., methotrexate, cyclophosphamides) have adverse side effects and many (e.g., L-asparaginase) are limited in their effectiveness by high dose requirements and restricted responsive tumor types. Thus, the evaluation of new, and hopefully better cancer chemotherapeutics, especially for those which may be effective in lower doses is a priority.
Thousands of compounds have been evaluated for in vitro anticancer activity. Only a few, however, have become clinically useful. Among these is one amino acid depleting enzyme, L-asparaginase. L-asparaginase has had success as an anti-cancer drug, especially in conjunction with other drugs, and in the treatment of acute lymphoblastic leukemia (ALL) and late stage cancers. L-asparaginase depletes the cells of the non-essential amino acid asparagine. L-asparaginase is advantageous in that it has been found to be relatively non-toxic for bone marrow. L-asparaginase, however, exhibits side effects which include allergic reactions (including anaphylaxis), hyperglycemia, liver dysfunction, coagulopathy, pancreatitis and CNS dysfunction. Evans et al. (Cancer (1990) 65:2624-2630); Hudson et al. (Cancer (1990) 65::2615-2618); Asselin et al. (Ca. Res. (1989) 49:4363-4368).
Those side effects related to L-asparginase being an immunogen (immune response) and those related to reduced protein synthesis as a consequence of amino acid depletion (e.g., coagulopathy and liver dysfunction) may be reasonably expected for arginine deiminase since it shares these properties. However, it can be expected that the immune response to arginine deiminase may be greatly reduced as an adverse side effect because the in vitro potency of this enzyme predicts lower doses will be required and thus lower side effects. While arginine deiminases convert the essential amino acid arginine to citrulline, it has not yet been determined that arginine depletion is the mechanism of tumor cell killing. It does, however, remain a likely theory.
It is known in the art that the covalent attachment of polyethylene glycol (PEG) to a protein blocks attack by degradative enzymes thereby inhibiting clearance from the circulation and converts immunogenic substances to nonimmunogenic derivatives. Brueck et al. (Der. Pharmacal. Ther. (1989) 12:200-204); Savoca et al. (Cancer Biochem. Biophys. (1984) 7:261-268); Abuchowski et al. (J. Biol. Chem. (1977) 252:3578-3582 and Cancer Treat. Rep. (1979) 63:1127-1132). U.S. Pat. No. 4,002,531 discloses a method of preparing PEG derivatives of enzymes having greater stability and greater retention of enzyme activity. U.S. Pat. No. 4,179,337 discloses the coupling of PEG to various polypeptides such as enzymes and peptide hormones such as insulin. U.S. Pat. Nos. 4,777,106 and 4,917,888 disclose the conjugation of PEG to an immunotoxin, .beta.-interferon and Interleukin 2. U.S. Pat. No. 4,791,192 discloses PEG-islet-activating protein (IAP) conjugates. The conjugates are described as having strong islet-activating activity and as producing less side effects than non-modified IAP. U.S. Pat. No. 4,847,325 discloses various methods of conjugating colony stimulating factor-1 (CSF-1) to PEG. The PEG-CSF-1 conjugate is described as being biologically active and as having an increased circulating half life. The conjugation of PEG to enzyme-type chemotherapeutics hold promise for an increased circulatory half-life and decreased immunogenicity of such chemotherapeutics.
Animal studies have suggested that arginase retard growth of some tumors in vivo. Bach et al. (Br. J. Ca. (1965) 9:379-384) demonstrated that bovine liver arginase inhibited the growth of Walker carcinoma cells in 31 of 40 (77%) rats for four days after the inoculation of tumor cells. However, survival time of animals was not significantly affected. Savoca et al. (Cancer Blochem. Biophys. (1984) 7:261-268) were able to both reduce tumor mass and extend the lives of rodents inoculated with tapir liver tumor cells and given bovine liver arginase modified with PEG to reduce immunogenicity and extend the circulating life of the enzyme. In these studies, 45% of the PEG-modified arginase remained in plasma after 24 hours, and 16% remained after 72 hours. In contrast, only 1% of non-modified arginase remained after 24 hours. The use of PEG-modified arginase was found to significantly increase survival of animals. Eighteen of 29 mice inoculated with tapir liver tumor cells survived greater than 60 days. Untreated animals survived a mean of 20 days. Fifteen of the 18 survivors were negative for gross or histological evidence of tumors.
Miyazaki et al. (Ca. Res. (1990) 50:4522-4527) identified tumor cell growth inhibitory activity in Rous sarcoma virus-transformed buffalo-rat liver (RSV-BRL) cells cultured in serum free conditioned media (SFCM) and discovered that the RSV-BRL cells were mycoplasma contaminated. It was determined that the growth inhibitory activity was due to mycoplasmal arginine deiminase. The enzyme was purified from the SFCM of the mycoplasma contaminated RSV-BRL cells and was shown to inhibit the growth of tumor cell lines in vitro. Although the species of the mycoplasmal contaminant was not identified, it was most likely one of the most common arginine deiminase producing tissue culture contaminants: M. arginini, M. orale, M. salivarium or M. fermentans. The enzyme and cytotoxic activity were absent after curing the cell culture of myeoplasmas. The arginine deiminase was effective against numerous human cancer cell lines including hepatomas, squamous cell carcinomas of the tongue and cervix, adenocarcinomas of the lung, nose and colon, glioblastoma, myeloma and melanoma. The mycoplasmal arginine deiminase was toxic for tumor cell lines at doses of 5 ng/ml. This was 1000-fold less than the previously reported minimal effective dose of bovine liver arginases.
The significantly increased specific activity of the mycoplasmal enzyme may be clinically advantageous. For example, lower doses may avoid serious immune reactions, such as anaphylaxis, which occur with the proteinaceous anti-neoplastic agent L-asparaginase. The extremely low in vitro effective dose of mycoplasmal arginine diaminase predicts lower in vivo doses and decreased risks of side effects.
The cloning and sequence analysis of arginine deiminase, but not expression of the protein, from one species of mycoplasma, M. arginini, has been reported by Ohno et al. (Infect. and Immun. (1990) 58:3788-3795) and Kondo et al. (Mol. Gen. Genet. (1990) 221:81-86).